Introgression of SUB1 aggravates the susceptibility of the popular rice cultivars Swarna and Savitri to stagnant flooding

Identification of the Sub1 gene for tolerance to flash flooding and its introgression into high-yielding rice cultivars are major targets in rice breeding for flood-prone rice agro-ecosystems for ensuring yield stability. However, knowledge is scant on the response of the modified genotypes under stagnant flooding (SF) to meet the challenge of finding a superior allele that may confer greater resilience to the plant under a stress-prone environment. In pursuance, we have tested the response of Sub1-introgression in two popular rice varieties, Swarna and Savitri to SF by comparing the biochemical factors in the control of flag leaf senescence and its primary production mechanisms of the parental lines’ versus Sub1-introgressed lines. The activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GR), and ascorbate peroxidase (APX) increased while various parameters of primary production like total chlorophyll content, stomatal conductance (gs), normalized difference vegetation index (NDVI) and photosynthetic activity (Pn) decreased progressively with passage of time in the flag leaf of the cultivars during the post-anthesis period and SF-treatment increased the enzyme activity while depressing primary production further. Introgression of Sub1 had no influence on these activities under control conditions but widened the margin of effects under SF. It was concluded that the functional ability of flag leaf in mega rice cultivars like Swarna and Savitri decreased significantly by SF because of an ethylene-mediated promotion of senescence of the flag leaf. The enhancement of antioxidant enzyme activity by SF could not sustain the stability of primary production in the flag leaf. The introgression of the Sub1 gene made the cultivars more vulnerable to SF because the gene induced overexpression of ethylene.


Result
Antioxidant enzyme activity. The activities of antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR), ascorbate peroxidase (APX), and catalase (CAT) in the flag leaf of both cultivars Swarna and Savitri and their Sub1-introgressed counterparts were low at day 7 post-anthesis and continued to rise temporally up to day 19 post anthesis both under control and stagnant flooding conditions (Figs. 1,2,3,4). The level of increase was higher under stagnant flooding (SF) in comparison to the control. The introgression of Sub1 into both Swarna and Savitri did not elicit any significant change in the activities of the enzymes over time in the control condition with some minor variations. However, the activities of the enzymes differed significantly between the cultivars with and without Sub1, when exposed to stagnant flooding. The enzyme activities remained consistently lower in the Sub1-introgressed Swarna and Savitri in comparison to their respective counterparts without Sub1 under stagnant flooding. General trends apart, the response of the two mega cultivars Swarna and Savitri to the introgression of Sub1-gene differed on some occasions for various anti-oxidant enzyme activity. In the control condition, SOD activity was lower, although not significantly, in the Sub1-introgressed genotype than its control counterpart in Savitri, but the activity was consistently higher in the Sub1-introgressed cultivar of Swarna compared to the control. This type of differential response between the two mega rice cultivars was not noticed in the measurement of activities of GR, APX, and CAT. The activities of these enzymes were similar for cultivars with and without Sub1 during the period of investigation.
Chlorophyll contents and photosynthetic-related activities. Contrary to the temporal increase in activities of the antioxidant enzymes in the post-anthesis period, the values P n, g s , NDVI, and total chlorophyll content of the flag leaf decreased consistently in the post-anthesis period in all the cultivars under both control and stagnant flooding conditions (Figs. 5,6,7,8). These values declined higher under stagnant flooding compared to the control condition, except on day 19 post-anthesis, where the values were higher than the control. The trend of decline was fastest in g s values while it was minimal for NDVI indices with the progress of time. www.nature.com/scientificreports/ Thus, the NDVI index remained relatively stable in the cultivars with the temporal change, in contrast to the other three indices, and was least affected by the stress. The introgression of the Sub1 locus into either Swarna or Savitri did not help maintain the stability of these crucial indices of biomass production. On all five occasions of sampling in the post-anthesis period, the values remained higher in the cultivars without Sub1 than that with Sub1.
Correlation between antioxidant enzyme and photosynthetic parameters. The activity of antioxidant enzymes (SOD, GR, APX, and CAT) showed a highly significant negative association with NDVI, g s , total chlorophyll content, and Pn of the flag leaf ( Table 1). The level of significance of APX with that of chlorophyll content and GR with NDVI was p < 0.01, whereas, for other parameters such as g s , NDVI and Pn, the correlation was significant at the level of p < 0.001. All other antioxidant enzymes showed a highly significant association (p < 0.001) with the photosynthetic parameters reported here.

Discussion
Rice grown in rainfed lowland areas frequently encounters submergence by flash flooding or stagnant flooding of different duration ranging from a week to months subject to the magnitude and pattern of rainfall. The susceptible cultivars succumb to submergence of 1-2 weeks duration 3,5 . Since long the Sub1 locus existing on rice chromosome 9 has been identified as the primary contributor of flooding tolerance 32 , it has enabled breeders to improve the submergence tolerance of popular rice cultivars of rainfed lowland areas like Swarna, by clever manipulation of the Sub1 locus through marker-assisted selection plant breeding method 19 . To augment yield benefit further, the breeding technique continues today to explore other important traits and pyramiding them with Sub1 10,33 . We noticed that semi-dwarf varieties like Swarna and Savitri with Sub1 QTL performed poorly and produced less yield under stagnant flooding as compared to their parental lines without Sub1 18 . Different yield parameters like tiller number, panicle weight, and total grain yield per unit area severely decreased under SF in Swarna-Sub1 and Savitri-Sub1as compared to their respective un-modified counterparts. Generation of the higher level of ethylene in the Sub1 varieties was found instrumental in depressing biomass and grain yield  18 . In the present dispensation, it might be possible that a retrograde action of intrinsic ethylene promoted leaf senescence under stagnant flooding and the effect was more discernible in the Sub1cultivars because of a compromise on the resilience mechanism. It is surmised that the ethylene-mediated enhancement of flag leaf senescence of the cultivars could be responsible for these effects on the biochemical parameters. In our previous work, it was noticed that the level of ethylene increased when both Swarna and Savitri cultivars were exposed to stagnant flooding, and introgression of Sub1 promoted ethylene release further under the stress 18 . The role of ethylene as a promoter of senescence in rice has been reported previously 34,35 . Ethylene was found responsible for chlorophyll loss, an increase of peroxidase enzyme activity, and a fall of PSII activity 34,35 , and results obtained in the present work reiterate the stance. The antioxidant enzymes expressed highly in the SF environment, more so in the Sub1-introgressed cultivars (Figs. 1, 2, 3, 4), but they could not preclude the adverse impact of stress on flag leaf primary production until day 19 after anthesis (Figs. 5,6,7,8) and the negative correlation between them was significant consistently (Table 1). Because Swarna and Savitri are popular for cultivation in shallow to medium-depth rainfed lowland ecosystems, the negative correlation as observed here could be generalized with respect to all other Sub 1-introgressed rice varieties, which are grown under this agroecosystem. It has been shown that in the gene cluster of Sub1, expression of Sub1A and Sub1C alleles are up-regulated by submergence and ethylene exposure whereas transcript levels of Sub1C increases also by gibberellic treatment 36 . However, Sub1A expression exhibits a negative association with gibberellic acid. It is possible that the expression of ethylene-responsive gene Sub1A was enhanced by the low oxygen environment (SF) in our experiment and upregulated activities of antioxidant enzyme superoxide dismutase, catalase, and ascorbate peroxidase (Figs. 1, 2, 3, 4) for scavenging the free radicals generated in the stress-prone environment 36 . Several instances are showing the critical role of ethylene in the production and accumulation of reactive oxygen species (ROS) in plants subjected to stress. Initiation and progression of leaf senescence are promoted by ethylene response factors in rice 37 . The ethylene response factors (ERFs) regulate ROS scavenging in plants 38 and they are intricately involved in coordinating stress response under an oxygendeficient environment 39 . Tiege et al. 40 showed that ethylene activates the MAPK cascade under salt stress and enhances the generation of reactive oxygen species. Ethylene promotes programmed cell death in sensitive plants whereas the interplay of ethylene signaling and reactive oxygen species stimulates the antioxidant defense system  5,6,7,8), which could be the causative factor responsible for the loss of biomass and grain yield 18 . In the stress-prone environment, the overproduction of reactive oxygen species significantly decreases the maximum quantum yield of PSII (Fv/Fm) and the decrease is closely associated with the increase of ion leakage and decrease of chlorophyll a/b ratio and chlorophyll contents of sensitive rice as against tolerant rice cultivars 43 . It is construed that the production and scavenging of reactive oxygen species confer differential sensitivity among the rice cultivars to stress. In our study, both Swarna and Savitri were SF-sensitive rice cultivars, as evident in the loss of chlorophylls and the corresponding reduction of P n activity (Figs. 5,6,7,8), while scavenging of reactive oxygen species was marginalized by a coincidental compromise on antioxidant enzyme activities under SF and introgression of Sub1 accentuated the marginalization further (Figs. 1, 2, 3, 4).
In rice plants, the flag leaf is the organ on which the stress-induced symptoms of the senescence syndromes are expressed and our observations on various physiological functions of the cultivars have most satisfactorily elucidated the stress effects. In stress-prone environments, a tolerant plant tends to suppress the overproduction of reactive oxygen species through scavenging these radicals by activation of antioxidant enzymes like SOD, GR, CAT, and APX 31 and failure of the enzyme activity amounts to a loss of longevity 44 . In the present investigation we observed that in all rice varieties with and without Sub1 under any environmental condition, the activities of these enzymes increased, and yet, the vital sign of life like photosynthesis and photosynthetic-associated parameters declined with the advancement of grain-filling in the post-anthesis period (Figs. 1, 2, 3, 4, 5, 6, 7, 8). It is surmised that the activities of the enzymes increased to neutralize the adverse influence of stress on primary production under a challenged environment and maintain the semblance of leaf stability. This assumption is corroborated by similar observations where overall leaf photosynthesis was found to decrease when the activities of antioxidant enzymes increased 44,45 .
In rice, delayed leaf senescence in the post-anthesis period facilitates grain filling by maintaining carbon assimilate supply longer through the stability of primary production 46 . Compared to low-yielding traditional www.nature.com/scientificreports/ rice, high-yielding improved cultivars have greater photosynthesis sustainability and maintain higher grain filling 2,44,47 . In the present study, photosynthesis was greater in all cultivars under control than SF up to 16 days post-anthesis (Fig. 6). NDVI values and chlorophyll contents were also greater during this time (Figs. 7, 8). NDVI index reiterates the reflection and absorption of light. Chlorophyll molecules absorb red light, whereas cellular structure reflects the incident light in the near-infrared band. So, low reflectance in the red band and greater infra-red reflectance is a healthy sign. Under stress conditions reflectance at the red band increases, whereas reflectance at the near-infrared band decreases. Maintaining greater NDVI indicates how healthy the plant is and this index remained most stable in our investigation. It is inferred that SF accentuated natural senescence-induced deleterious effects on Pn and associated photosynthetic parameters accounting for depression on grain yield 18

Conclusion
In summation Swarna and Savitri are naturally stagnation-susceptible varieties. The authors reported that introgression of Sub1 promoted overexpression of ethylene activity, which further aggravated the susceptibility of these two varieties under water stagnation. Thus, they recommended not to introgress Sub1 into stagnant susceptible genotypes. However, no information or data was given on what might happen when Sub1 is introgressed in the water stagnation tolerant genotypes.

Materials and methods
Plant material. Two popular rice (Oryza Sativa L.) cultivars Swarna (Vasista × Mashuri) and Savitri (Pankaj × Jagannath) and their SUB1near isogenic lines (NILs), Swarna-Sub1 and Savitri-Sub1 were used in the experiment. These cultivars were released by ICAR-National Rice Research Institute, Cuttack for cultivation in shallow to medium-depth rainfed lowland ecosystems.
Treatments and experimental design. The experiment was conducted in alluvial sandy clay loam soil of the Mahanadi River delta (pH -6.7, organic 0.89%, total N 0.01%, available P 22 kg ha −1 , and available K 125 kg ha −1 ) at National Rice Research Institute, Cuttack India (20.5° N, 86° E and 23.5 m above sea level) during the wet seasons of 2014 with three replications under factorial randomized block design. The date of sowing was 21st June and after one month, seedlings were transplanted on the date 21st of July. Inorganic fertilizers N:P:K was applied at 60:30:30 kg ha −1 . Phosphorous as single super phosphate and K as muriate of potash were applied as basal doses, whereas N as urea was applied in two split doses, 50% after 7 days of transplanting and the rest 50% three days before the imposition of SF. The experiment was incubated in two side-by-side field tanks (length × breadth × height: 40 m × 8 m × 0.8 m), in which one was used as control where water depth varied from 2 to 10 cm. The other field tank was used for stagnant flooding (SF). 30 days old seedlings were transplanted in the experimental tanks @ single seedling hill −1 in a plot (5 m × 3 m). Each tank had twelve plots, hill-to-hill space was 15 cm and line-to-line distance was 20 cm. SF was imposed after one month of transplanting by a gradual increase of water level @ 10 cm day −1 . Approximately 40-50 cm water depth was maintained up to the anthesis stage and discontinued thereafter. The water level decreased gradually, yet 5-10 cm standing water stayed in the SF treatment tank at harvest. www.nature.com/scientificreports/ Chlorophyll measurement. The flag leaf of the main shoot was sampled at three-day intervals between days 7 and 19 post anthesis and frozen in liquid nitrogen and stored in a freezer (− 80 °C) until estimation of enzyme assay. One hundred mg of finely chopped fresh flag leaf was used to estimate the total chlorophyll content. The leaves were placed in a capped vial, which contained 25 mL of cold (4 °C) acetone. The vial with the sample was placed in a refrigerator (4 °C) for 28 h for extraction of chlorophyll 49 . The chlorophyll was measured spectrophotometrically by taking optical density at 663.6 and 646.6 nm following the procedure of Porra 50 . The chlorophyll contents were calculated using the following equation. The chlorophyll content was calculated as mg g −1 fresh weight basis.

Normalized difference vegetation index (NDVI). NDVI was measured using an NDVI meter (Plant
Pen model NDVI 310, Photon Systems Instruments, Drásov, Czech Republic). NDVI index is calculated by measuring plant reflectance in the visible and near-infrared wavelengths. As NDVI is a ratio, it has no unit. NDVI 310 uses the following formula to calculate the NDVI index. Riboflavin was added at the end. The reaction was monitored in the presence of two 40 V fluorescent lamps for 20 min. One unit of SOD activity was defined as the amount of enzyme that caused 50% inhibition of the rate of NBT reduction at 560 nm by using a spectrophotometer (model SL 164, Elico, Hyderabad, India). APX (EC 1.11.1.11) was assayed according to Nakano and Asada 53 by monitoring the rate of ascorbate oxidation at 290 nm by using extinction coefficient = 2.8 m mol cm −1 . The reaction mixture contained 50 mM potassium phosphate buffer of pH 7.0, 0.1 mM EDTA, 100 mM H 2 O 2, and 0.5 mM ascorbic acid and enzyme aliquot.
CAT (EC 1.11.1.6) activity was assayed according to the method of Cakmak and Marschner 54 . The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 10 mM H 2 O 2, and the enzyme extract. The decomposition of H 2 O 2 was monitored at 240 nm.
GR (EC 1.6.4.2) activity was assayed according to the method of Foyer and Halliwell 55 by following the decrease in absorbance at 340 nm caused by NADPH oxidation by using extinction coefficient = 6.2 mM cm −1 .
Measurement of photosynthetic gas exchange parameters. CO 2 photosynthetic gas exchange rate (Pn) was measured under ambient environmental conditions using a portable open-system photosynthetic gas analyzer (model TPS1, PP Systems International, Amesbury, USA). Three separate measurements from each replication were done using a fully grown flag leaf. The total numbers of measurements were nine for each treatment. The selected flag leaf was kept inside the chamber under natural irradiance until a stable reading was observed. Different gas exchange parameters like CO 2 photosynthetic rate (µmol CO 2 m −2 s −1 ) and stomatal conductance (mmol H 2 O m −2 s −1 ) are presented here.
Statistical analysis. Differences between the various parameters were assessed through comparison by ANOVA using the software CROPSTAT (International Rice Research Institute, Manila, Philippines). Means were compared by the least significant differences provided the F test was significant. Associations among different traits were examined by simple correlation and regression analysis using the same software.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.